Bee-less honey

Patent metadata

Field	Value
Publication number	US-12247231-B2
Application number	US-202117503506-A
Country	US
Kind code	B2
Filing date	Oct 18, 2021
Priority date	Oct 18, 2020
Publication date	Mar 11, 2025
Grant date	Mar 11, 2025

Abstract

Systems comprising a first polynucleotide comprising a regulatory element operatively linked to an open reading frame encoding an invertase enzyme and a second polynucleotide comprising a repressible regulatory element operatively linked to an open reading frame encoding a glucose oxidase enzyme wherein the repressible element inhibits transcription in response to hydrogen peroxide are provided. Cells and compositions comprising a system of the invention, as well as methods using the system of the invention, are also provided, as is artificial honey.

First claim

The invention claimed is:

1. A kit comprising:
i. a first expression vector polynucleotide comprising at least one first promoter operatively linked to a first open reading frame, wherein said first open reading frame encodes a signal peptide and an invertase enzyme, wherein said invertase enzyme is encoded by a sequence comprising SEQ ID NO: 4 or SEQ ID NO: 5 or a sequence homologous thereto that converts one molecule of sucrose into one molecule of glucose and one molecule of fructose; and
ii. a second expression vector polynucleotide comprising a repressible promoter operatively linked to a second open reading frame, wherein said second open reading frame encodes a signal peptide and a glucose oxidase enzyme, wherein said glucose oxidase enzyme is encoded by a sequence comprising SEQ ID NO: 6 or 7 or a sequence homologous thereto that converts one glucose molecule into one molecule of glucono-1,5-lactone and one molecule of hydrogen peroxide, and wherein said repressible regulatory element inhibits transcription of said second open reading frame in response to hydrogen peroxide.

2. The system kit of claim 1 , wherein
wherein said signal peptide is operatively linked to said enzyme to induce secretion of said enzyme by a cell.

3. The kit of claim 1 , wherein said promoter is a bacterial promoter, said first promoter is a constitutive promoter or both.

4. The kit of claim 1 , wherein said repressible promoter inhibits transcription of said second open reading frame and said inhibition is proportional to hydrogen peroxide levels.

5. The kit of claim 4 , further comprising a hydrogen peroxide sensitive promoter operatively linked to a third reading frame, wherein said third reading frame encodes a repressor of said repressible promoter and wherein said hydrogen peroxide sensitive promoter induces transcription of said third reading frame in response to hydrogen peroxide.

6. The kit of claim 5 , wherein said hydrogen peroxide sensitive regulatory element is a Pkat promoter, said repressor is LacI, said repressible regulatory element is a Plac promoter, or a combination thereof.

7. The kit of claim 5 , further comprising a fourth polynucleotide molecule comprising a regulatory element operatively linked to a fourth open reading frame, wherein said fourth open reading frame encodes a catalase enzyme and wherein said catalase is functional to convert hydrogen peroxide to water and oxygen.

8. A cell comprising the first expression vector polynucleotide and second expression vector polynucleotide of the kit of claim 1 .

9. The cell of claim 8 , wherein said cell is a bacterial cell, is a B. subtilis cell, comprises endogenous expression of a catalase enzyme, or a combination thereof.

10. The cell of claim 8 , wherein said polynucleotides are expression vectors functional within said cell, said signal peptides are functional within said cells to induce secretion of proteins to which said signal peptides are operatively linked or both.

11. A composition comprising the cell of claim 8 cultured in a solution comprising sucrose.

12. The composition of claim 11 , wherein said solution comprises between 20-50% sucrose.

13. The composition of claim 11 , wherein said cell and growth media for said cell devoid of sucrose are within a semipermeable container and said semipermeable container is within said solution comprising sucrose and wherein said semipermeable container is configured to allow invertase and glucose oxidase to diffuse out of said growth media into said solution comprising sucrose and not allow glucose to diffuse from said media comprising glucose into said growth media.

14. A composition comprising the cell of claim 8 and growth media for said cell devoid of sucrose within a semipermeable container, wherein said semipermeable container is configured to allow invertase and glucose oxidase to diffuse out of said growth media and not allow glucose to diffuse into said growth media.

15. A method for producing bee-less honey, the method comprising culturing the cell of claim 8 in a solution comprising sucrose, thereby producing bee-less honey.

16. The method of claim 15 , wherein
a. said solution comprising sucrose comprises between 20-50% sucrose;
b. said culturing comprises culturing the composition of claim 15 within said solution comprising sucrose;
c. said culturing further comprising adding sucrose to said solution if sucrose concentration in said solution is below a predetermined threshold; or
d. a combination thereof.

17. An artificial honey solution produced by a method of claim 15 .

18. An artificial honey solution, comprising at least 40% fructose, at least 30% glucose, at most 10% sucrose and at most 20% water.

19. The kit of claim 1 , being for use in transfecting or infecting a bacterial cell.

20. The kit of claim 1 , wherein said invertase is encoded by a sequence comprising SEQ ID NO: 4 or SEQ ID NO: 5 and said glucose oxidase is encoded by a sequence comprising SEQ ID NO: 6 or SEQ ID NO: 7.

Assignees

• Technion Res & Dev Foundation

Inventors

• Amit Roee
• Levi Shira
• Tannenzapf Lidya
• Zur Yehonatan
• Brajzblat Ilan
• Licht Assaf
• Warsha Ofri
• Dror Mai
• Ben Meir Dor
• Haim Lior
• Awwad Zeinat
• Litver Nir
• Davidov Tzila
• Abrahami Pachuk Liron
• Eden Noa
• Tibi Hanriet
• Atar Orna

Classifications

• C12N9/0006

  acting on CH-OH groups as donors (1.1) · CPC title

• C12Y101/0301

  Pyranose oxidase (1.1.3.10) · CPC title

• C12Y302/01026

  Beta-fructofuranosidase (3.2.1.26), i.e. invertase · CPC title

• C13K3/00

  Invert sugar; Separation of glucose or fructose from invert sugar · CPC title

• C12P19/02

  Monosaccharides (2-ketogulonic acid C12P7/60) · CPC title